5. Cryogenic optical nanoscopy

Recent developments in single-molecule fluorescent microscopy made it possible to circumvent the optical resolution limit of approximately 200 nm given by Abbes law. By imaging individual molecules sequentially and assembling an image from a large number of thus obtained molecular positions, these techniques achieve resolutions between 10 – 100 nm. This resolution lies within the dimension of structures found in biological systems, such as multiprotein-complexes of cells.

We are investigating a family of GTPases called septins that are involved in a number of neurodegenerative diseases and several forms of cancer. Several, differentially expressed septins form tissue-specific complexes. EM and X-ray crystallographic studies revealed that theses complexes are non-polar rods with a length of approximately 30 nm. The assembly of these rods into higher-order structures is essential for septin function. However, it remains unclear, how exactly the septin rods are arranged within these higher-order structures.

We aim to use photoactivation localization microscopy (PALM) to investigate the arrangement of septin complexes within higher-order structures. As known from the resolved X-ray structure of the human septin complex, septin7 resides at its ends. We will label septin7 with photoswitchable or photoconvertable fluorophores that can be used for PALM. With a 30 nm length of septin complexes and a 20 nm resolution in PALM, we hypothesize that it will be possible to reveal the orientation of septin rods within the higher-order structure.

In parallel, we will establish single molecule microscopy at cryogenic temperatures. Since fluorophores at this temperature show higher photostability this results in a higher number of photons per molecule. Since the localization precision is roughly dependent on the number of photons we expect to achieve a better resolution of single molecules.

In future work, we aim to develop cryo-superresolution microscopy further for the application in cells. In respect to this, we expect that we can reveal the supramolecular organization of septin complexes in cells. The development of diverse single molecule microscopy techniques in the last years provides new possibilities for the investigation of macromolecular biological studies. By employing PALM and super-resolution microcopy at cryogenic temperatures we expect to reveal the orientation of septin complexes within the higher-order structure. In addition, we want to investigate the order of individual septin isoforms in the complex. These results will be of high interest in the septin research field, since the higher-order structure is essential for septin function.